Alcoholics go through multiple detoxifications with each episode being more severe and the likelihood of relapse higher with each episode. In addition, cues associated with alcohol drinking can enhance 'craving' and precipitate relapse drinking. The overall goals of this project are to determine neuronal mechanisms underlying cue-induced ethanol (EtOH)-seeking behavior and the effects that repeated deprivation cycles have on these mechanisms. The overall hypothesis is that increased activities of dopamine (DA) and glutamate (Glu) pathways within the meso-cortico-limbic system underlie cue-induced EtOH-seeking behavior and the activities of these pathways are further enhanced with repeated deprivation cycles. A Pavlovian Spontaneous Recovery (PSR) test will be used to measure expression of EtOH-seeking behavior in alcohol-preferring (P) rats following prolonged abstinence. Repeated 2-week cycles of abstinence and operant ethanol access will be used to evaluate the effects of repeated deprivations on cue-induced EtOH-seeking behavior. No-net-flux (NNF) and conventional in vivo microdialysis techniques will be used to examine the involvement of DA, Glu and GABA systems in EtOH-seeking behavior, and changes in these systems associated with distinct cues and repeated deprivations. Microinjection procedures will be used to identify pathways and receptors involved in regulating EtOH-seeking behavior. Aim 1 will determine the interactions of odor cues and repeated deprivations on expression of EtOH-seeking behavior and transmitter release. Aim 2 will assess the effects of repeated deprivations on basal neurotransmission of DA, Glu and GABA within the meso-limbic system that persists in the absence of EtOH. Aim 3 will use microinjection techniques to assess the involvement of different receptors in regulating cue-induced EtOH-seeking behavior. Overall, this project will provide important information on neuronal mechanisms involved in regulating cue-induced EtOH-seeking behavior and the impact of repeated deprivations on these neuronal systems.